Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

Summary

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Abstract

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

Introduction

The virulence of many Gram-negative bacterial pathogens depends on specialized secretion systems to hijack eukaryotic host cells. Bacteria use these secretion systems to inject bacterial virulence proteins (effectors) into the host cell to modulate a variety of cellular and biochemical activities. The study of effector proteins has not only provided remarkable insight into fundamental aspects of host/pathogen interactions but also into the basic biology of eukaryotic cells¹. Modulation of host cell apoptosis has been shown to be an important virulence mechanism for many intracellular pathogens, and a number of effector proteins modulating apoptosis have been identified^2-9. However, their precise molecular mechanisms of activity remain elusive in many cases.

Apoptosis, a form of programmed cell death, plays an important role in immune responses to infection¹⁰. Two main pathways leading to apoptosis have been identified: targeting the mitochondria (intrinsic apoptosis) or direct transduction of the signal via cell death receptors at the plasma membrane (extrinsic apoptosis). The intrinsic or mitochondria-mediated cell death pathway is triggered by intracellular signals and involves the activation of Bax and Bak, two pro-apoptotic members of the Bcl-2 family. This family is composed of pro- and anti-apoptotic regulator proteins that control cell death^11-14. Activation of apoptosis leads to oligomerization of Bax and Bak followed by subsequent permeabilization of the mitochondrial outer membrane, resulting in cytochrome C release into the cytoplasm. Cytochrome C release initiates activation of the effector caspases 3 and 7 through activation of caspase 9 in the apoptosome¹⁵. This leads to proteolysis of selected substrates that, among others, results in the exposure of phosphatidylserine on the cell surface¹⁶ and frees a dedicated DNase that fragments chromatin^17,18.

In order to determine where within the apoptotic cascade an individual effector protein interferes, an inducible expression system was employed¹⁹. Regulatory systems for conditional expression of transgenes have been an invaluable tool in analyzing a protein’s function within the cell or its importance for tissue, organ and organism development, as well as during initiation, progression and maintenance of disease^20-23. Typically, inducible control systems, such as the Tet system²⁴ employed here, form an artificial transcription unit (see Figure 1). One component is an artificially engineered transcription factor called tTA (tetracycline-dependent transcription activator), formed by fusion of the bacterial transcription repressor TetR²⁵ to a mammalian protein domain that mediates transcriptional activation or silencing ^24,26. The second component is a hybrid promoter, termed TRE (tetracycline-responsive element), consisting of a eukaryotic minimal promoter, containing at least a TATA-box and a transcription initiation site, joined to multiple repeats of the cognate DNA-binding site for TetR, tetO^24,25. The third component is the natural ligand of TetR, tetracycline or one of its derivatives, such as anhydrotetracycline or doxycycline²⁵. Upon ligand addition to the culture medium, TetR loses its affinity for tetO and dissociates from the TRE. As a result, transcription of the target gene is abolished. Transgene expression can, thus, be tightly controlled in a time- and dose-dependent manner in both cell culture and in animals^20,23,24. With tTA, transgene expression occurs constitutively, except in the presence of a tetracycline. This can be a disadvantage in the study of cytotoxic or oncogenic proteins because tetracycline first has to be removed from the system, before transgene expression occurs and the target protein‘s effects on the cell can be monitored. This can be time-consuming and is not always complete, especially in transgenic animals²⁷. To address this limitation, a TetR mutant with an inverse response to the presence of doxycycline was used to generate a new transcription factor, rtTA (reverse tTA)²⁸. It only binds to the TRE and, concomitantly, activates transcription in the presence of doxycycline. Residual leakiness of the system, i.e., transgene expression in the absence of TRE-bound transcription factor, originating either (i) from position effects at a genomic integration site, (ii) from the TRE itself²⁹, or (iii) from non-specific binding of tTA/rtTA²⁸, was addressed by introducing an additional transcriptional silencer, termed tTS (tetracycline-dependent transcriptional silencer)³⁰ to the system. It forms a dual regulator network together with rtTA (see Figure 1). In the absence of doxycycline, tTS binds to TRE and actively shuts down any remaining transcription. In the presence of doxycycline, tTS dissociates from TRE and rtTA binds simultaneously inducing expression of the target gene. This additional layer of stringency is often necessary to express highly active cytotoxic proteins^31-34.

Using this tightly controlled dual-regulator system, the apoptotic cascade can be initiated at a defined step allowing analysis of whether the given effector protein can interfere with apoptosis induction. This method can not only be used to study the anti-apoptotic activity of bacterial effector proteins but also for the inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Protocol

1. Generation of Stable Cell Lines Expressing the Protein of Interest

1. Prepare media by adding heat-inactivated FCS and 1% Penicillin/Streptomycin to commercially available Dulbecco´s Modified Eagle Medium (DMEM) supplemented with GlutaMAX-I, Pyruvate, and 4.5 g/L D-Glucose.
2. Cultivate HEK293 cells in media at 37 °C in 5% CO₂. Sub-cultivate cells every third day. Remove the media and resuspend the cells in 15 ml fresh media. Pipette 1 ml into a new 75 cm² cell culture flask and add 14 ml media.
3. Analyze the cell number by using a hemocytometer.
4. Seed HEK293 cells in a 6-well plate at a density of 2 x 10⁵ cells per well and incubate for 24 hr.
5. For transfection use the protocol provided by the manufacturer of the transfection reagent. Transfect cells with pEGFP-C2 or pEGFP-C2-CaeB using the transfection reagent. Prior to use, warm up the transfection reagent and the media required to RT. Use a 3:1 ratio of transfection reagent to DNA.
   1. In detail, pipette 1.5 µl of transfection reagent directly into 50 µl of serum-free DMEM medium. For complex formation, pipette 0.5 µg of DNA encoding GFP or GFP-CaeB to the transfection reagent-containing mixture and incubate for 15 min at RT.
   2. Transfect cells by adding the reaction mixture in a drop-wise manner and incubate at 37 °C in 5% CO₂ for 24 hr.
6. Add 1 mg/ml geneticin for selection of GFP-positive clones at 37 °C in 5% CO₂.
7. After 6 days, isolate single cells by flow cytometry sorting in a 96-well plate harboring medium and HEK293 supernatant in a 1:1 ratio. Generate HEK293 supernatant from cultured confluent HEK293 cells that were centrifuged for 15 min at 4,966 x g.
8. On the following day, replace culture medium with medium containing 1.5 mg/ml geneticin. Change media once a week. If the cells form a confluent monolayer, transfer them to a 24-well plate. Sub-cultivate cells twice a week in a ratio of 1:5 in media containing 1.5 mg/ml geneticin. Finally, analyze the percentage of GFP-positive cells by immunoblot analysis and flow cytometry.

2. Analysis of Stable Cell Lines by Immunoblot Analysis

1. Remove the supernatant and wash the adhered cells once with warm PBS. Resuspend the cells in 100 µl of 2x Laemmli sample buffer, heat for 5 min at 95 °C and store at -20 °C.
2. Load approximately 4 x 10⁴ cells per sample on a 12% SDS-PAGE gel. Additionally, load 6 µl of a commercial protein ladder as a molecular weight marker. Run gels in a gel electrophoresis system under a constant voltage of 180 V for 45-60 min with 1x Laemmli running buffer.
3. Blot proteins onto PVDF membranes (pore size of 0.45 µm) at a constant voltage of 16 V for 60 min using a Trans-Blot SD Semi-Dry Transfer Cell.
4. Block membranes in 10 ml of 5% nonfat dry milk in PBS/0.05% Tween 20 (MPT) for 1 hr at RT.
5. Dilute 4 µl of anti-GFP antibody in 4 ml of MPT and incubate membranes O/N at 4 °C.
6. Perform three 10 min washing steps with 10 ml of PBS/0.05% Tween 20.
7. Incubate membranes with 0.8 µl of horseradish peroxidase-conjugated secondary antibodies in 4 ml of MPT.
8. After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 (as described in 2.6) and detect horseradish peroxidase activity with a commercial kit according to the manufacturer’s protocol. Apply standard equipment for film development to visualize protein bands.

3. Analyze the Cells Using a Flow Cytometer.

1. Resuspend cells in PBS. Use HEK293 cells as negative control to adjust forward scatter (FSC) and side scatter (SSC) so that the cells are on scale. Draw a gate on living cells by excluding cell doublets, aggregates and cell debris.
2. Adjust the photomultiplier tube (PMT) gain so that the unstained cells are on the far left of the histogram for the FL1 channel (488 nm argon laser).
3. To analyze the fluorescence intensity of HEK293-GFP and HEK293-GFP-CaeB cells open the histogram of the FL1 channel and acquire 10,000 events on the gated population.
4. Analyze the data using commercial FACS analysis software.

4. Transfection of the Stable Cell Line with the Inducible Expression Vector System

1. Seed HEK293 cells stably expressing GFP or GFP-CaeB in a 12-well plate at a density of 1 x 10⁵ cells/well.
2. 19 hr post-seeding, co-transfect the cells with regulator plasmid and response plasmid either encoding Bax or activated caspase 3.
   1. Co-transfect the stable HEK293 cells with 100 ng of pWHE125-P regulator plasmid (Figure 2) and 100 ng of the response plasmids pWHE655-hBax or pWHE655-revCasp3 (Figure 3) and 0.4 µl of polyethylenimine.
   2. Prepare the DNA and polyethylenimine transfection reagent each in 75 µl of Opti-MEM medium and incubate for exactly 5 min at RT. For complex formation, incubate both mixtures together for 15 min at RT.
3. Add the polyethylenimine/DNA solution drop-wise to the cells and incubate at 37 °C in 5% CO₂.

5. Induction of Apoptosis

1. 5 hr post-transfection, induce expression of the pro-apoptotic proteins by addition of 1 µg/ml doxycycline to the culture medium. Incubate cells for 18 hr at 37 °C in 5% CO₂.

6. Analysis of Host Cell Apoptosis by Immunoblot Analysis

1. Inspect cells prior to harvesting under the light microscope to check for cell death induction. Apoptotic cells are detectible by the presence of apoptotic bodies surrounding the dying cell.
2. Remove the supernatant and wash the adhered cells once with warm PBS. Resuspend the cells in 100 µl of 2x Laemmli sample buffer, heat for 5 min at 95 °C and store at -20 °C.
3. Load approximately 4 x 10⁴ cells per sample on a 12% SDS-PAGE gel. Additionally, load 6 µl of a commercial protein ladder as a molecular weight marker. Run gels in a gel electrophoresis system under a constant voltage of 180 V for 45-60 min with 1x Laemmli running buffer.
4. Blot proteins onto PVDF membranes (pore size of 0.45 µm) at a constant voltage of 16 V for 60 min using a Trans-Blot SD Semi-Dry Transfer Cell.
5. Block membranes in 10 ml of 5% nonfat dry milk in PBS/0.05% Tween 20 (MPT) for 1 hr at RT.
6. Dilute 4 µl of anti-cleaved poly ADP-ribose polymerase (PARP) antibody in 4 ml of MPT and incubate O/N at 4 °C.
7. Perform three 10 min washing steps with 10 ml of PBS/0.05% Tween 20.
8. Incubate membranes with 0.8 µl horseradish peroxidase-conjugated secondary antibodies diluted in 4 ml MPT.
9. After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 (as described in 6.7) and detect horseradish peroxidase activity with a commercial kit according to manufacturer’s protocol. Apply standard equipment for film development to visualize protein bands.
10. Remove bound antibodies by 30 min incubation at RT with 7 ml Western Blot Stripping Buffer.
11. After three washes with PBS/0.05% Tween 20 (as described in 6.7), probe the membranes with 4 µl of anti-actin antibody in 4 ml of MPT O/N at 4 °C.
12. After three washing steps with MPT (as described in 6.7), incubate the membranes with 0.8 µl of horseradish peroxidase-conjugated secondary antibodies diluted in 4 ml of MPT.
13. After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 (as described in 6.7 and detect horseradish peroxidase activity with a commercial kit according to the manufacturer’s protocol. Apply standard equipment for film development to visualize protein bands.
    Note: All incubation steps were performed on a plate shaker for the indicated time and temperature.

Results

First, HEK293 cell lines stably expressing the protein of interest (CaeB) as a GFP-fusion protein were established. As a control, HEK293 cell lines stably expressing GFP were also generated. Expression of GFP and GFP-CaeB was verified by immunoblot analysis. The representative immunoblot (Figure 4A) demonstrates stable and clearly detectable expression of GFP and GFP-CaeB. However, this assay cannot determine whether all cells express GFP or GFP-CaeB. Therefore, the stably transfected HEK293 cell lines w...

Discussion

Many pathogenic bacteria harbor secretion systems to secrete or translocate bacterial effector proteins into the host cell. These effector proteins have the capacity to modulate processes and pathways in the host cell, allowing the bacteria to survive and replicate within their respective intracellular niche. Understanding the biochemical activities and the molecular mechanisms of the effector proteins will help towards a better understanding of pathogenicity and may help to develop new therapeutic tools to combat diseas...

Materials

Name	Company	Catalog Number	Comments
DMEM	life technologies	31966-021
FCS	Biochrom	S0115
Pen/Strep	life technologies	15140-122
OptiMEM	life technologies	51985
X-tremeGENE 9	Roche	6365752001
Geneticin	Roth	CP11.3
Polyethylenimine	Polyscienes	23966
Doxycycline	Sigma Aldrich	D9891
Mini-PROTEAN Tetra Cell	Bio-Rad	165-8000EDU
Trans-Blot SD Semi-Dry Transfer Cell	Bio-Rad	170-3940
PageRuler Prestained Protein Ladder	Thermo Scientific	26616
PVDF membrane	Millipore	IPVH00010
anti-GFP	life technologies	A6455
anti-cleaved PARP	BD Bioscience	611038
anti-actin	Sigma Aldrich	A2066
Mouse IgG (H+L)-HRPO	Dianova	111-035-062
Rabbit IgG (H+L)-HRPO	Dianova	111-035-045
ECL Western Blotting Substrate	Thermo Scientific	32106
Restore Plus Western Blot Stripping Buffer	Thermo Scientific	46428